Angiogenesis is the process by which new blood vessels are formed, with accompanying increased blood circulation. The field of angiogenesis has been a favorite for research and investigation for over one hundred years. See, e.g. Virchau, R., Die Krankhaftern Geshwultste, Hirshwald, Berlin (1863); Thiersch, C., Die Haut Mit Atlas, Leipzig (1865); (significance of the interaction between host vasculature and survival and growth of solid malignant tumors observed). Interest has been fueled by the observation that angiogenic factors are found, in trace amounts, in normal tissue. See, e.g., D'Amore et al., PNAS 78:3068-3072 (1981); Kissun, et al., Br. J. Ophthalmol. 66:165-169 (1982); (retinal tissue); DeCarvallho, et al., Angiology 34:231-243 (1983); (activated lymphocytes and macrophages); Frederick, et al., Science 224:289-390 (1984); (human follicular fluid); Burgos, Eur. J. Clin. Invest. 13:289-296 (1983); (amniochorion and placenta); Castellot, et al PNAS 79:5597-5601 (1982); (culture medium of 3T3 cells). The trace amounts of angiogenic factors observed in these tissues do not, however, show any angiogenic activity other than in normal growth and development of tissues and organs. Similarly, angiogenic factors have been observed in tissues of pathological origin. See, e.g., Weiss, et al., Br. J. Cancer 40:493-496 (1979); Fencelau, et al., J. Biol. Chem. 256:9605-9611 (1981), McAslau, et al., Exp. Cell Res. 119:181-190 (1979); (tumor cells); Kumar, et al., Lancet 2:364-367 (1983); Brown, et al., Lancet 1:682-685 (1980) (synovial fluid of arthritis patients); Hill, et al., Experientia 39:583-585 (1983) (vitreous material of diabetics); Banda, et al., PNAS 79:7773-7777 (1982) (wound fluid).
Goldsmith et al, (1984) JAMA 252: 2034-2036 is the first report of an angiogenic factor which shows activity beyond normal growth and development, and is available in large quantities. The factor was found in chloroform/methanol fractionates of feline omenta (CMFr). See the co-pending application Ser. No. 642,624, filed Aug. 20, 1984, entitled "Angiogenesis Factor and Method for Producing Angiogenesis," of Catsimpoolas and Goldsmith. This application is incorporated by reference herein.
It has further been found that the crude lipid extract of Goldsmith et al. (1984), Supra may be purified into various fractions which possess angiogenic properties far above those observed in the CMFr.
Additionally, it has been found that commercially available gangliosides such as gangliosides derived from brain tissue and other lipid containing compounds also possess angiogenic properties. Further, new compositions of known lipid containing compounds may be formed which also possess angiogenic properties.
The discovery of lipid containing compounds which possess angiogenic properties is relatively new to the art. Previously attention has been focused on proteinaceous angiogenic factors. See, e.g., Kumar et al., Lancet II:364-367 (1983) (proteinaceous factors from 300 to 10.sup.5 daltons); Kissun, et al., supra (proteinaceous factors up to 70 kd); Banda, et al., supra (proteins of about 2-14 kd); Burgos, et al., supra, (protein complexes of from 100-200 kd). It has now been unexpectedly shown that compositions containing lipid containing molecules, such as gangliosides, glycolipids, ceramides, cerebrosides, phospholipids, sphingosides, and so forth, exhibit enhanced angiogenic activity as disclosed in co-pending U.S. patent application Ser. No. 782,724, filed Oct. 1, 1985 and incorporated by reference herein.
Angiogenic lipid containing compounds are known to increase vascular perfusion in wound areas of test animals as described in co-pending U.S. patent application Ser. No. 805,206 filed on Dec. 4, 1985 and incorporated herein by reference. Also see Goldsmith, et al., Surgery 162:579-583 (Jun. 1986) and JAMA 252: at 2035 (1984).
The lipid containing compounds have also been found to exhibit unexpected results when used to treat angina and myocardial infarctions as disclosed in U.S. patent application, Ser. No. 811,375, filed on Dec. 20, 1985 and incorporated by reference herein. Tests have shown, for example, that vascularization, neovascularization and vascular collateralization are accelerated. This is a surprising result since lipid materials are regarded more as atherosclerotic agents than vascularizing agents in heart tissue.
Lipids are displaced from homogenized cell membranes, or other complexes involving proteins by detergent molecules which render the proteins "soluble" in aqueous media as discussed in co-pending U.S. patent application, Ser. No. 805,206, filed on Dec. 4, 1985 and incorporated by reference herein. Also see Colombo, M. I. et al. Biol. Cell 54:73 (1985) or Chuang, D. M., et al. J. Cyclic Nucleotide Protein Phosphor Res. (U.S.) 10:281 (1985) and Wall, D. A. et J. Cell. Biol. 101:2104 (1985). One theory proposed, but in no way intended to limit the inventors to just this theory is that amphiphilic compounds or detergents, used to "solubilize" the lipid components from the cell surface may indirectly induce angiogenesis since angiogenic lipids, such as phospholipids, glycolipids, and gangliosides, are found on biological membranes. See co-pending U.S. application, Ser. No. 782,724, supra.
Since most amphiphilic compounds aggregate in aqueous media to form micelles above certain concentrations called the "critical micellar concentration" (CMC), they have been found to be effective in dissolving lipids from biological membranes mainly by forming mixed micelles with lipids. See, e.g., Lichtenberg, et al., Biochim. Biophys. Acta, 737: 285-304 (1983); (structural and kinetic aspects of solubilization of phospholipids by detergents); Helenius, et al., Biochim. Biophys. Acta, 415:29-79 (1975); (solubilization of membranes by detergents); and Hjelmeland, L. M., Methods in Enzymology, 124:135-164 (1986); (design and synthesis of detergents for membrane biochemistry).
The art supports some use of amphililic compounds in biological situations. We refer to the work of Pitha and Szente in the Journal of Pharmaceutical Sciences 73:240 (1984) wherein derivatives of digitonin are formed which lower amphiphilic compound toxicity. In that work CH.sub.2 OH groups of digitonin were converted to CH.sub.2 OH.sub.2 CHOHCH.sub.3 or to CH.sub.2 OCH.sub.2 CHOHCH.sub.2 O(CH.sub.2).sub.4 OCH.sub.2 CHOHCH.sub.2 OH groups. We note the toxicity varies greatly from species to species and in mode of use, Pitha and Szente ibid P. 242. Pat. No. 4,546,097 issued Oct. 8, 1985 to Josef Pitha discusses mycoplasma suppression in culture and increased solubilization of lipophilic drugs. However there is no angiogenic effect taught therein.
The angiogenic results of the invention are therefore surprising since as reported in the above paper by Pitha and Szente, digitonin has been known for some inflammatory and hemolytic effects.
The work of Segel et al. (1977) Biochem. Pharm. 26:643 discusses protection against the hemolytic effect of digitonin using a specific saponin, glycyrrhizin (P. 644). Digitonin has been used to prevent hypercholesterolemia in monkeys Malinow, M. R., et al. (1978) Amer. J. Clin. Nutrition 31:814. Cholesterol adsorption regulation in humans with digitonin is the subject of a Malinow et al. U.S. Pat. No. 4,242,502. Generally digitonin forms insoluble complexes with cholesterol.
Digitonin was tolerated by both rats and monkeys in that work (see P. 817). We also note that a mixture of digitonin glucosides comprise the commercial material. Therefore the angiogenic effect may be expected for all congeners therein as detailed by Y. M. Yang et al. (1986) Biomedical and Environmental Mass Spectroscopy 13:439.
It is thus an object of this invention to describe specific amphiphilic compounds and their use to enhance angiogenesis and blood perfusion properties in mammals.
It has now been unexpectedly shown that particular amphiphilic compounds have angiogenic properties as discussed infra. The solubilization of lipid components by amphiphilic compounds from the surface of normal cells may indirectly induce angiogenesis.